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A RP-HPLC method for the determination of tea catechins
CANCER LETTERS
ELSEVIER
Cancer Letters 114 (1997) 171-172
A RP-HPLC method for the determination of tea catechins Santosh Kh~khar~>~, Dini Venemaa, Peter C.H. Hollmana,*, Matthijs Dekkerb, Wim Jongenb ‘DLO-State
Institute
for Quality Control of Agricultural Products (RIKILT-DLO), Bornsesteeg NL-6708 PD Wageningen, The Netherlands bDepartment of Food Science, PO Box 6700 EV Wageningen, The Netherlands
45,
Abstract An HPLC method with gradient elution for the quantification of catechins ((-)-epigallocatechin (EGC), (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechingallate (EGCg) and (-)-epicatechingallate (ECg)) in tea was developed. The method was used to determine catechins in black tea, green tea and oolong tea. Sample preparation was simple because only filtration and adjustment of pH was required. The levels of catechins in different teas varied between <5 and 287 (EGC), 17 and 94 (EC), 18 and 408 (EGCg), and 8 and 85 mg/l (ECg). (+)-Catechin was not present in any of these teas. 0 1997 Elsevier Science Ireland Ltd. Keywords:
Tea; Catechins; HPLC; Analysis; Antioxidant
1. Introduction
2. Materials
Flavanols (catechins) in tea are a major source of antioxidants in the human diet. A number of health claims concerning various flavonoids have been made from epidemiological and experimental animal studies. They seem to play an important role in reducing the risk of the formation of a number of ageing diseases like cancer and coronary heart diseases. Analysis of the catechin content of different types and brands of tea is therefore important for comparing these teas for their possible health promoting potential.
2.1. HPLC
-* Correspondingauthor.
The HPLC system consisted of an automatic injector, a Merck Hitachi L6200 and a L6000 pump to form a high pressure gradient, and an Inertsil-ODS-2 (GL Sciences Inc.) column (150 x 4.0 mm, 5 pm) protected by an Opti-Guard PR Cl8 Violet A guard column (Optimize Technologies, Inc.) both placed in a column oven set at 3O”C, and a UV detector set at 278 run. The separation of major catechins was carried out by using 5% acetonitrile (eluent A) and 25% acetonitrile (eluent B) in phosphate buffer (0.025 M, pH 2.4). The gradient used was: O-5 min, 15% B; 5-20 min, linear gradient 15-80% B; 20-23 min, 80% B; 23-25 min, 15% B. The flow rate was 1.0 ml/min. Catechins
used for calibration were obtained from Sigma ((+>-
0304-3835/97/$17.00 0 1997Elsevier ScienceIrelandLtd. All rights reserved PZZ SO304-3835(97)04653-3
and methods
172 Table
I
The content” Tea
of catechins
in different
-
Rlack tecr Ceylon (N1.i Yule (India) PG-Tips (UK) Gwrn fC(d China Japan oolon~ il’li China
teas (m&l)
EGC -__,
._-___.____”
EC ,... --._-_-
0! -.y <5”
3 31 40
I b.7 2x7
,i; 04
17 ----.-----.
i7
- ._--
---.-_.---
EGCg
11s
-
-
- ----
EC’@ --.--
7
.-. -... -- .-.--------
To1al
--
‘ii.
“Means of 1 -3 determinations. ?imit of detection.
catechin (C). (-)-epicatechin (EC)) and APIN, UK ((-)-epigallocatechin (EGC), (-)-epigallocatechingallate (EGCg) and (-)-epicatechingallate (ECg)).
3. Tea extract Teas were prepared by extracting 1 g of dry matter with 100 ml of freshly boiled water (100°C) for 5 min including shaking for 30 s. After the extraction the pH was adjusted to 3.2 with citric acid. The extract was diluted five times with water and liltered through a 0.45 pm filter. 2.1. Results und discussion Several columns, Hypersil-ODS (Chrompack), Nova-Pak (Waters Millipore) and Inertsil-ODS-2 were tested with isocratic and gradient elutions. Inertsil-ODS-2 with gradient elution gave the best separation. The achievable resolution of (+)-catechin and caffeine was not sufficient to quantify small amounts of (+)-catechin (<25 mg/l). However, no (+)-catechin was detected (limit of detection 10 mg/l) in any of the tea samples.The method was usedto determinecatechins in black, green and oolong tea from different origins (Table 1). As expected, green tea contained higher amounts of catechins than black and oolong tea. Green tea from Japan contained even higher levels of catechinsthan green tea from China. The black tea, Ceylon with Pickwick brand name, sold in The Netherlands contained much higher amountsof catechins than the black tea from India,
and was in fact similar to the green tea from China. EGCg and EGC were the predominant catechins in most teas. Green as well as black tea in this study did not contain (+)-catechin (
4. Conclusions
The gradient method developed for the determinalion of catechinsis suitable for all kinds of tea and is faster than other published methods.Samplepreparation is simple becausesampleclean up was not necessary.
References ill
Balentine, D.A. ( 1992) Manufacturing and chemistry of tea. In: Phenolic Compounds in Food and their Effects on Iiealth 1. S07. p. 102. Editors: C.-T. Ho, C.Y. Lee and M.-T. Huang. American Chemical Society, Washington DC. PI Graham, H.N. (1992) Green tea composition. consumption and polyphenol chemistry, Prev. Med., 21. 334. 131 Ham, Y.. (1994). Prophylactic kmctions of tea polyphenols. In: Food Phytochemicals for Cancer Prevention II, p. 3J Editors: C.-T. Ho, T. Osawa. M.-T. Huang and R.T. Rosen. American Chemical Society. Washington DC.
ELSEVIER
Cancer Letters 114 (1997) 171-172
A RP-HPLC method for the determination of tea catechins Santosh Kh~khar~>~, Dini Venemaa, Peter C.H. Hollmana,*, Matthijs Dekkerb, Wim Jongenb ‘DLO-State
Institute
for Quality Control of Agricultural Products (RIKILT-DLO), Bornsesteeg NL-6708 PD Wageningen, The Netherlands bDepartment of Food Science, PO Box 6700 EV Wageningen, The Netherlands
45,
Abstract An HPLC method with gradient elution for the quantification of catechins ((-)-epigallocatechin (EGC), (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechingallate (EGCg) and (-)-epicatechingallate (ECg)) in tea was developed. The method was used to determine catechins in black tea, green tea and oolong tea. Sample preparation was simple because only filtration and adjustment of pH was required. The levels of catechins in different teas varied between <5 and 287 (EGC), 17 and 94 (EC), 18 and 408 (EGCg), and 8 and 85 mg/l (ECg). (+)-Catechin was not present in any of these teas. 0 1997 Elsevier Science Ireland Ltd. Keywords:
Tea; Catechins; HPLC; Analysis; Antioxidant
1. Introduction
2. Materials
Flavanols (catechins) in tea are a major source of antioxidants in the human diet. A number of health claims concerning various flavonoids have been made from epidemiological and experimental animal studies. They seem to play an important role in reducing the risk of the formation of a number of ageing diseases like cancer and coronary heart diseases. Analysis of the catechin content of different types and brands of tea is therefore important for comparing these teas for their possible health promoting potential.
2.1. HPLC
-* Correspondingauthor.
The HPLC system consisted of an automatic injector, a Merck Hitachi L6200 and a L6000 pump to form a high pressure gradient, and an Inertsil-ODS-2 (GL Sciences Inc.) column (150 x 4.0 mm, 5 pm) protected by an Opti-Guard PR Cl8 Violet A guard column (Optimize Technologies, Inc.) both placed in a column oven set at 3O”C, and a UV detector set at 278 run. The separation of major catechins was carried out by using 5% acetonitrile (eluent A) and 25% acetonitrile (eluent B) in phosphate buffer (0.025 M, pH 2.4). The gradient used was: O-5 min, 15% B; 5-20 min, linear gradient 15-80% B; 20-23 min, 80% B; 23-25 min, 15% B. The flow rate was 1.0 ml/min. Catechins
used for calibration were obtained from Sigma ((+>-
0304-3835/97/$17.00 0 1997Elsevier ScienceIrelandLtd. All rights reserved PZZ SO304-3835(97)04653-3
and methods
172 Table
I
The content” Tea
of catechins
in different
-
Rlack tecr Ceylon (N1.i Yule (India) PG-Tips (UK) Gwrn fC(d China Japan oolon~ il’li China
teas (m&l)
EGC -__,
._-___.____”
EC ,... --._-_-
0! -.y <5”
3 31 40
I b.7 2x7
,i; 04
17 ----.-----.
i7
- ._--
---.-_.---
EGCg
11s
-
-
- ----
EC’@ --.--
7
.-. -... -- .-.--------
To1al
--
‘ii.
“Means of 1 -3 determinations. ?imit of detection.
catechin (C). (-)-epicatechin (EC)) and APIN, UK ((-)-epigallocatechin (EGC), (-)-epigallocatechingallate (EGCg) and (-)-epicatechingallate (ECg)).
3. Tea extract Teas were prepared by extracting 1 g of dry matter with 100 ml of freshly boiled water (100°C) for 5 min including shaking for 30 s. After the extraction the pH was adjusted to 3.2 with citric acid. The extract was diluted five times with water and liltered through a 0.45 pm filter. 2.1. Results und discussion Several columns, Hypersil-ODS (Chrompack), Nova-Pak (Waters Millipore) and Inertsil-ODS-2 were tested with isocratic and gradient elutions. Inertsil-ODS-2 with gradient elution gave the best separation. The achievable resolution of (+)-catechin and caffeine was not sufficient to quantify small amounts of (+)-catechin (<25 mg/l). However, no (+)-catechin was detected (limit of detection 10 mg/l) in any of the tea samples.The method was usedto determinecatechins in black, green and oolong tea from different origins (Table 1). As expected, green tea contained higher amounts of catechins than black and oolong tea. Green tea from Japan contained even higher levels of catechinsthan green tea from China. The black tea, Ceylon with Pickwick brand name, sold in The Netherlands contained much higher amountsof catechins than the black tea from India,
and was in fact similar to the green tea from China. EGCg and EGC were the predominant catechins in most teas. Green as well as black tea in this study did not contain (+)-catechin (
4. Conclusions
The gradient method developed for the determinalion of catechinsis suitable for all kinds of tea and is faster than other published methods.Samplepreparation is simple becausesampleclean up was not necessary.
References ill
Balentine, D.A. ( 1992) Manufacturing and chemistry of tea. In: Phenolic Compounds in Food and their Effects on Iiealth 1. S07. p. 102. Editors: C.-T. Ho, C.Y. Lee and M.-T. Huang. American Chemical Society, Washington DC. PI Graham, H.N. (1992) Green tea composition. consumption and polyphenol chemistry, Prev. Med., 21. 334. 131 Ham, Y.. (1994). Prophylactic kmctions of tea polyphenols. In: Food Phytochemicals for Cancer Prevention II, p. 3J Editors: C.-T. Ho, T. Osawa. M.-T. Huang and R.T. Rosen. American Chemical Society. Washington DC.